Lipopolysaccharide as Receptor for Rhizobium Phage 1P

Abstract

INTRODUCTIONThere are only a few papers concerning the sites of phage adsorption in Rhizobium. Barnet & Vincent (1970) suggested that the receptor site for phages 7,7cr, and 8 was associated with the somatic antigen of Rhizobium. Atkins & Hayes (1972) found alterations in lipopolysaccharide (LPS) and lipoprotein (LP) of Rhizobium trifolii mutants that adsorbed phage poorly.To elucidate the attachment site of phage IP to Rhizobium, we studied cell walls and their LPS preparations as phage receptors. METHODSBacterial andphage strains. Rhizobium trifolii strains 24SM, HR, 1 5 2 4~~ and XSM were used in these experiments. Mutants HR and 15, resistant to phage IP, were derived from 2 4~~. The former was obtained by U.V. treatment, whereas the latter was a spontaneous mutant isolated by selection with phage IP. Phage IP propagated on R. trijiolii 2 4 s~ was employed in assays of receptor activity of cell walls and LPS. It does not require divalent ions for its attachment to bacteria (Staniewski, 1968).Media. Bacteria were grown in medium 4, containing (g/l glass-distilled water) : K2HP0,, 3-6; MgS04.7Hz0, 0.5; NaCl, 0.5; ferric ammonium citrate, 0.05; Casamino acids, 3.0; glucose, 10.0; pH 7-6. Bacetylation of cell walls and LPS by NaOH and NH40H. Cell walls and LPS (5 mg/ml) Preparation of cell walls, LPS and polysaccharides (PS).were treated with 0.1 M-NaOH or 2.5 M-NH,OH at 37 "C for I h. The mixture was chilled, neutralized with 0.1 M-HCl and centrifuged. The pellet was washed three times and ly ophilized. LPS oxidation with NaI04. LPS was oxidized according to a modification of the procedure of Foster, Davies & Crumpton (1958). One ml of LPS (10 mg/ml) was mixed with 0.5 ml of 2 % (w/v) NaIO, and incubated at 20 "C for 12 min. Excess NaIO, was decomposed by ethylene glycol. After 20 min, 0.5 ml of I % (w/v) NaBH, was added. Excess NaBH, was then decomposed by acetic acid (0.2 ml) and dialysed owmight against distilled water.Inactivation of phage IP. Cell walls of LPS, at a concentration of I to 50 ,ug/ml in tris-HC1 buffer pH 7.4, were mixed with an equal volume of phage IP containing 6 x IO* plaque forming units (p.f.u.). The mixture was incubated at 37 "C without shaking. At intervals, samples were removed and tested for phage survival using R. trifolii 24SM as the indicator culture. 24Y I C 90Downloaded from www.microbiologyresearch.org by

Host-Symbiont Interactions

“…Since many phage receptors of enteric bacteria reside in the LPS (6,18,32,38,39), phage lysis of Rhizobium was studied with the idea that this may be an additional probe for examining the similarity of the LPS of the various Rhizobium strains. However, there is no obvious correlation between phage lysis pattern and LPS composition or immunochemistry (see Tables I and II, and Fig.…”

“…The partial compositions of the LPS from several strains of R. trifolii and R. leguminosarum have been reported (14,15,21,22,38). Immunochemical studies of the LPS of the two R. trqfolii strains have suggested that these LPS are strain-specific antigenic determinants of these bacteria (14).…”

Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis

“…Rhizobium lipid A appears to be much like that of Escherichia coli and Salmonella species (32, 35,36). As with the enterobacteria, most of the variability of rhizobial LPS resides in the carbohydrate moiety, which may vary as greatly between strains of the same species as between species (4,47). Like LPS of rough mutants of enterobacteria (31) and unlike other rhizobial LPS, R. meliloti LPS contains a high percentage of KDO (47), which, together with other evidence, suggests that it may not have much 0 antigen.…”

Isolation and characterization of a bacteriophage specific for the lipopolysaccharide of rough derivatives of Pseudomonas aeruginosa strain PAO

“…Feige and Strim (17) have carried out phage inactivation studies in buffer and broth and have found that neutralization is usually superior in broth. The PhI5o value for 4PLS27 with AK-44 LPS is 0.08 jig/ml, which compares very favorably with the lowest PhI50 values obtained with smoothspecific phages in P. aeruginosa (30,40) and with LPS-specific phages in other genera (23,43,44,52,68).…”